Retinal degeneration is the leading cause of blindness in the United States and other first world countries characterized by loss of retinal photoreceptor cells. The neural retina is subject to a number of degenerative conditions, including retinitis pigmentosa, age-related macular degeneration, and glaucoma. Although there are a number of sources of progenitors for regeneration in non-mammalian vertebrates, these are greatly reduced or absent in the adult mammalian retina. By contrast, recent reports show that retinal progenitor cells can be derived from mouse embryonic stem (ES) cells and may provide an alternative to adult derived retinal stem cells. In other regions of the central nervous system, the transplantation of neurons derived from ES cells has led to some promising results. However, the use of ES cells requires the destruction of the embryos and creates ethical issues and concerns. Recent reports have demonstrated that somatic skin cells in mice and in humans can be induced to pluripotency with the addition of genes delivered using retroviruses. Nonetheless, these approaches require the retroviral infections and induce tumorigenecity, which makes them inadequate for therapeutic use. Recently, two publications, both in Nature, have shown that in mice spermatogonial stem cells (SSCs) can reprogram to embryonic stem (ES) cells without the addition of genes or retroviruses. However, to date, there is no evidence showing the pluripotency of adult human spermatogonial stem cells. Lately, we have demonstrated that adult human SSCs can be reprogrammed to pluripotency spontaneously in appropriate culture condition, without genomic modification and retroviral infection. We have successfully isolated and cultured the adult human spermatogonial stem cells for the first time and under defined culture conditions, we have reprogrammed the SSCs to pluripotent stem cells capable of differentiating into all three germ layers. We have induced the differentiation of various cell lineages including neuronal precursors expressing neuronal specific markers, such as, nestin, neurofilament, synaptophysin and dopaminergic receptor 2 (Drd2). Additionally, we have generated a Stra8-EGFP transgenic mouse line that specifically expresses GFP in the SSCs, a source for obtaining a pure population of adult mouse SSCs. Based upon our preliminary results in human and mouse, our goals are to: (1) optimize the long-term expansion and reprogramming of human and mouse SSCs into ES-like cells by optimizing the culture conditions, verify the stability of normal karyotype, gene expression and the tumorigenicity or teratomas formation, (2) investigate differentiation capacity of SSC-derived ES-like cells into neural cells and particularly retinal neurons, make them proliferate and possess the ability to propagate in vitro and to become functional neurons. Our future goal after these two-year pilot studies is to inject the human and mouse SSC-derived neuro-retinal progenitor cells into the retina of a mouse model of retinal degeneration and to analyze the capacity of these cells to regenerate and repopulate the retina. This approach is of paramount importance since human testicular biopsies may allow the cell-based, autologous organ therapy without the ethical and immunological problems associated with human embryonic stem cells, in the absence of genetic modification and retroviral infection. PUBLIC HEALTH RELEVANCE: Retinal degeneration is the leading cause of blindness in the United States and other first world countries characterized by loss of retinal photoreceptor cells. Recent reports show that retinal progenitor cells can be derived from mouse embryonic stem (ES) cells and may provide an alternative to adult derived retinal stem cells. However, the use of ES cells requires the destruction of the embryos and creates ethical issues and concerns. Recently, we have demonstrated that adult human spermatogonial stem cells (SSCs) can be reprogrammed to pluripotency spontaneously, using appropriate culture media, without genomic modification and retroviral infection. We have reprogrammed the SSCs to pluripotent stem cells capable of differentiating into all three germ layers. We have induced the differentiation of various cell lineages including neuronal precursors expressing neuronal specific markers, such as, nestin, neurofilament, synaptophysin and dopaminergic receptor 2 (Drd2).This approach is of paramount importance since human testicular biopsies may allow the cell-based, autologous organ therapy without the ethical and immunological problems associated with human embryonic stem cells, in the absence of genetic modification and retroviral infection. In this proposal we will examine the biology, the plasticity and therapeutic potential of human spermatogonial stem cells.